In spite of great experimental and theoretical effort the mechanism of protein folding still remains one of the fundamental unsolved problems of molecular biology. This is largely due to the difficulty in obtaining specific structural information on short-lived intermediate states on the folding pathway. High-resolution NMR can provide the desired structural resolution, if transient conformations can be labeled or trapped in stable form. We propose to use two-dimensional 1H-NMR in combination with rapid mixing techniques and hydrogen exchange for the structural and kinetic characterization of the folding pathway in cytochrome c. Structural intermediates at different stages of refolding will be studied by allowing hydrogen exchange to compete against structure acquisition. The extent and location of proton label trapped during refolding will be analysed by 2D J-correlated spectroscopy, making use of numerous specifically assigned NH-CAlphaH cross peaks (so far 60 amide protons have been assigned). One- and two-dimensional magnetization transfer methods will further be employed for the kinetic characterization of the unfolding transition under equilibrium conditions and to study the properties of the unfolded state. Finally the importance of the heme ligands for stabilizing the cytochrome c structure and their role in folding will be investigated by 2D NMR and NH-exchange characterization of derivatives with altered ligation. In all these experiments detailed structural information will be obtained by virtue of the capability to observe numerous individual protons at known positions within the cytochrome c structure.